ARTICLE IN PRESS 2 Intensity of aggressive interactions modulates testosterone 3

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ARTICLE IN PRESS
Physiology & Behavior xx (2004) xxx – xxx
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Intensity of aggressive interactions modulates testosterone
in male marmosets
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Corinna N. Rossa,*, Jeffrey A. Frencha,b, Kimberly J. Pateraa
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Department of Biological Sciences, University of Nebraska at Omaha, United States
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Department of Psychology, University of Nebraska at Omaha, United States
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Received 17 January 2003; received in revised form 24 March 2004; accepted 30 August 2004
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Abstract
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Androgen is associated with the expression of male-typical behavior, including aggressive behavior, but high levels of androgen may be
incompatible with other behavioral systems, such as paternal care. In a variety of species of birds that display paternal care, testosterone (T)
levels in males are maintained at low levels, and these levels rise only in response to direct agonistic challenges. This idea has not been
thoroughly studied in mammals with biparental care, and we exposed male marmosets (Callithrix kuhlii), a monogamous and biparental
primate to aggressive interactions with unfamiliar intruders. Urinary levels of T and cortisol (CORT) were monitored prior to and following
these interactions. Baseline T was not correlated with variation in aggression in either residents or intruders, and CORT was not affected by
the encounters. However, males responded to an encounter with male intruders with changes in T that correlated with the level of aggression
displayed by the resident male during the trial. Encounters with male intruders that elicited high frequencies of aggressive displays by the
male resident were associated with increased T 2–6 h and 24 h following the encounter, and encounters that had few aggressive displays
resulted in no change or a decrease in T concentrations. Intruders did not demonstrate a significant relationship between T and aggression.
Thus, the magnitude of the hormonal response is dependent on the intensity of aggression during a male–male encounter, suggesting that
elevated androgens are likely to be a consequence, rather than a cause, of aggressive interactions in marmosets.
D 2004 Published by Elsevier Inc.
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Keywords: Marmoset; Callitrichid; Testosterone; Aggression; Intruder
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1. Introduction
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Testosterone (T) affects the development and function of
reproductive characteristics in males, including gametogenesis, sexual behavior, aggressive behavior, and maturation of secondary sexual characteristics [1–3]. These
features all contribute to male reproductive success. At the
same time, however, maintaining high concentrations of T
may be associated with considerable costs for males. High
concentrations of T are associated with decreased immune
function, decreased energy reserves, and decreased paternal
investment [4] (cf. Refs. [5,6]). In species whose social
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* Corresponding author. Department of Biology, 348 Manter Hall,
University of Nebraska at Lincoln, Lincoln, NE 68588-0118, United States.
Tel.: +1 402 472 3999; fax: +1 402 472 2083.
E-mail address: [email protected] (C.N. Ross).
systems are characterized by a high variance in male
reproductive success, the costs associated with elevated T
may be outweighed by the potential that T-mediated traits
lead to greater success in siring offspring. In polygynous
avian species with little or no paternal care, T titers are
maintained at high levels throughout the breeding season to
facilitate aggressive interactions for territory and mate
acquisition [4,7–9]. However, in monogamous species that
show paternal care, androgen levels are high only during the
period of territory and mate acquisition, and are low
throughout the rest of the year. In spite of the low levels,
however, males retain the ability to respond to territorial or
aggressive challenges from other males with transient
elevations in T, presumably to facilitate aggression during
these intruder challenges [4,7–9].
Studies of aggression and testosterone in primates have
focused on species that form large troops containing
0031-9384/$ - see front matter D 2004 Published by Elsevier Inc.
doi:10.1016/j.physbeh.2004.08.036
PHB-07378; No of Pages 9
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ARTICLE IN PRESS
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2. Methods
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throughout the encounter. We expected the intensity of
agonistic encounters to be greatest in male–male encounters.
If the intruder elicits an aggressive response from the
resident (i.e.: poses a territorial challenge to the resident),
then exposure to a male intruder should be associated with
increases in the resident male’s T titers. Since the reaction to
the agonistic encounter may be different for an intruder
animal than for the resident, we also monitored the
intruder’s behavioral and endocrine responses to these trials.
Finally, because agonistic encounters serve as potent
stressors in a wide variety of species [10,12,25,26], we
monitored changes in cortisol (CORT) across the phases of
the experimental encounters.
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2.1. Subjects
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2.1.1. Residents
Five pairs of Wied’s black tufted-ear marmosets (C.
kuhlii), consisting of an adult breeding male and his mate,
served as resident subjects for the study. The animals were
housed at the University of Nebraska at Omaha Callitrichid
Research Facility. The animals were maintained in pairs
and small family groups in cages measuring at least
1.20.92.4 m. The cages contained natural branches, a
nest tube, a feeding platform, and enrichment devices.
Visual access between groups was limited, but olfactory
and auditory contact was available with two to three other
family groups. For further details of animal husbandry and
housing, see Ref. [27].
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2.1.2. Intruders
Twenty marmosets were used as intruder subjects in our
observations. Thirteen marmosets (six males and seven
females) were adult intruders (N24 months) and were
breeding adults in their own social groups. Seven intruders
(four males and three females) served as juvenile intruders.
Juveniles were between the ages of 6 months and 2 years of
age, and resided in a social group that contained older adult
parents.
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2.2. Procedure
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2.2.1. Intruder trials
The residents were exposed and habituated to the
intruder cage before any testing began by placing the empty
intruder cage in each home cage for three 8-h time periods
prior to the first test. We avoided testing groups with
females in the third trimester of pregnancy or with nursing
infants. The resident pair and intruder were first observed
during a 10-min pre-trial period. The intruders were
removed from their home cage by coaxing them into a
small transport cage attached to their home cage. From the
transport cage the animals were transferred to an intruder
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multiple males and females. Male aggression in these
species is often a result of intraspecific intrasexual conflict.
These conflicts are often due to changes in and attempts to
maintain hierarchical status. Studies of these species have
found that males express very high levels of testosterone
and high levels of aggression towards other males during an
encounter [10–13]. Although basal levels of T may be low
in these species due to overall stable social systems, a
conflict results in drastic changes in T concentrations. In
fact, the concentrations of T are thought to be linked to the
outcome of the interaction, with the dominant bwinnerQ
showing increases in T, while the subordinate bloserQ shows
drastic decreases in T following the interaction [10–12].
While previous studies of primates have focused upon
species that have multiple males with varying levels of
paternal care, to date no study has examined the hormonal
response of primate males to an aggressive encounter in a
species that shows high levels of male investment in the
offspring.
We tested whether T concentrations in male black tuftedear marmosets (Callithrix kuhlii) predicted aggressive
behavior in encounters with unfamiliar males, and whether
T levels were altered as a consequence of engaging in an
aggressive interaction with a stranger. Marmosets are
socially monogamous primates, and have high levels of
paternal care [14–19]. The infants are fully dependent on
caregivers for 5–6 weeks after birth, and males begin
carrying infants at 1 week of age, and are the primary
carriers from 3 weeks of age until weaning [20]. Nunes et al.
[20,21] have demonstrated that T levels are lowest in males
during the period of maximal offspring care, that T is higher
in males with less paternal experience, and that T levels are
higher in males that carry infants at low rates. Previous
behavioral studies have revealed that callitrichids are
differentially aggressive towards intruders [22–24]. Specifically, male black tufted-ear marmosets responded with the
highest frequency of aggressive displays when exposed to
an unfamiliar intruder, they displayed less aggression
towards a familiar male, and even less to a female intruder
[24]. Male–male aggression in callitrichids appears to differ
from other primates previously studied [10–13], because
callitrichid conflict appears to be driven by territorial
maintenance rather than intragroup conflict to maintain
hierarchical status. Male hormonal responses to male–male
aggression during territorial encounters in marmosets have
not been studied. Although we may predict from other
primate studies that an aggressive encounter would be
associated with an increase in T, it is unknown whether the
hormonal response will be dependent on the sex of the
intruder, or on the status of the encounter (i.e.: a win versus
a loss).
We used an intruder paradigm [22–24] to test the
behavioral and endocrine responses of males to aggressive
encounters with conspecifics. Residents were exposed to
encounters with adult and juvenile intruders of both sexes,
and the intensity of agonistic interactions was monitored
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2.2.3. Control intruder
The intruder was placed in the intruder cage and then
placed in a novel empty cage, the size of standard family
housing. This trial was used to control for non-encounter
related handling effects. Observations were made as
described for the experimental trial. The animal was then
released into its home cage and observed.
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2.2.2. Control resident
An empty intruder cage was placed in the resident cage
and observations were performed in the same manner as
described above. After the 30-min trial period the intruder
cage was removed from the room and a 10-min post
observation was conducted.
TE
Method
State of resident
Moving
Rest
Feeding
Grooming
Playing
Instantaneous
Instantaneous
Instantaneous
Instantaneous
Instantaneous
Description
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Social interactions
Allogrooming
Contact
Near
Copulating
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The
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The
focal
focal
focal
focal
focal
animal
animal
animal
animal
animal
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is
is
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traveling to a new position
not traveling
consuming food or water
scratching its own body
in apparent play behavior alone, or with others, usually includes chase behavior
Grooming or being groomed by the mate
The resident pair is in physical contact
The focal animal is within 10 cm of its mate
The focal animal is engaged in mating behavior
Proximity to cage
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Instantaneous
Within 10 cm of the intruder cage
Agonistic responses
Erh–erh
Long call
Twitter
Scent marking
Genital display
Attack
Piloerection
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Vocalization which is a low gutteral sound typically accompanied by attack behavior
Vocalization which is used as a contact call, long in duration and high in pitch
Multi-syllable vocalization that follows a long call
A genital rub on branches or other surfaces
Exposing genital area by lifting the tail
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The focal animal’s hair is extended from body
Intruder demeanor
Neutral–attentive
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Submissive
Agonistic
Instantaneous
Instantaneous
Instantaneous
Instantaneous
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Instantaneous
Instantaneous
Instantaneous
All occurrences
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male juvenile, and 3 weeks separated all trials with an
animal, whether the animal served as a resident or an
intruder. Additionally, at least 2 days separated all trials in
the colony in order to prevent a general disturbance to the
colony. Exposure of residents to a male and female intruder
was alternated. Control trials were randomly scheduled
among experimental trials while leaving 3 weeks between
each use of an animal, these trials served as control for
both behavioral responses to novel situations, as well as a
control for the daily circadian rhythms of hormones to be
assayed.
Table 1
Definitions of behaviors collected during the intruder trials for both the residents and intruders (as defined in French et al., 1995; Schaffner and French, 1997)
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cage (604040 cm), constructed of wire mesh. The
intruder cage eliminated the possibility of physical contact
between residents and intruders, while still providing good
visual contact between interacting marmosets. After the
intruders were transferred to the intruder cage they were
allowed to acclimate to the cage for 5 min in an empty
room. After this period the intruder was placed in the home
cage of the resident pair to be tested.
Two observers were required for the intruder trial, one to
record the behavior of the intruder and the resident of the
same sex, and one to record the behavior of the other
resident. Inter-observer reliability was at least 90% for all
trials, as was determined by comparing affiliative behavior
of the pair during the trial. Observations occurred at 20-s
intervals for 30 min, and were recorded using Observer
3.0R on a laptop computer. Recorded patterns of agonistic
and aggressive behavior toward the intruder, affiliative
behavior within a pair, and general activity of both the
resident and intruder were recorded during each observation
as described in Table 1 [24,28]. Following the intruder trial,
the intruder was released back into its home cage and the
behavior of both residents and intruder was monitored for an
additional 10 min. All intruder encounter trials started
between 0800 and 0900 h.
Each resident pair was presented with two different
adult males, two adult females, and one female and one
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occurrences
occurrences
occurrences
occurrences
occurrences
occurrences
occurrences
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The
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intruder
intruder
intruder
intruder
follows movement but makes no response
is non-responsive to the actions of the resident
is cowering and vocalizing in low tones
actively attacks and chases the resident
ARTICLE IN PRESS
2.3.1. Testosterone
T concentrations were measured in samples using an
enzyme immunoassay adapted for C. kuhlii outlined in
Refs. [21,30]. Urine samples (10 Al) were diluted in 1000
Al phosphate-buffered saline (PBS) and incubated for 18 h
at 37 8C after the addition of 25 Al h-glucuronidase (Type
H2, Sigma, 2700 Fishman units). Samples were then
extracted with 5 ml anhydrous diethyl ether, evaporated to
dryness, and reconstituted with 2 ml PBS. Extraction
efficiency was monitored by the external recovery of 3H-T
added to extraction tubes and was 99.9% (n=7). The
microtitre plates (Nunc-Immuno Plate MaxiSorp F96)
were coated with rabbit anti-T: BSA (#5/98) diluted
1:15,000 in bicarbonate coating buffer. Standards were
diluted with PBS and ranged from 1000 to 1.95 pg (ICN
Diagnostics). The steroid conjugate (horseradish peroxidase) T-HRP (#11/98) was diluted 1:15,000 in PBS;
recovery of the standards was 104.7F10.4%. Intra-assay
coefficients of variation for the high and low control pools
were 4.33% and 4.16%, respectively (n=9). The interassay coefficients of variation for the high and low control
pools were 13.84% and 12.57%, respectively (n=9).
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2.3.2. Cortisol
CORT concentrations were measured in all urine
samples using an enzyme immunoassay developed and
validated for use in C. kuhlii, as previously described in
Refs. [31,32]. Urine samples were diluted 1:6400 with
deionized water. Microtitre plates were coated with 50 Al
per well of antibody (R4866, raised against a steroid
bovine albumin (BSA) in rabbit, diluted to 1:16,000 in EIA
phosphate buffer). Standards were diluted in water and
ranged from 1000 to 1.95 pg (Sigma). The steroid
conjugate (horseradish peroxidase) was diluted in EIA
phosphate buffer to a dilution of 1:30,000. Recovery of all
standards (range 1.95–1000 pg) added to quality control
pools was 101F2%. The intra-assay coefficients of
variation for medium and low concentration pools were
4.46% and 3.47%, respectively (n=20). The inter-assay
coefficients of variation for the medium and low concentration pools were 14.29% and 17.75%, respectively
(n=20).
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2.4.1. Behavioral analysis
Since residents experienced two separate intruder
encounters with adult intruders, the replicate trials with
adult intruders were collapsed into a composite score for
each intruder sex. All behavioral data were standardized to
a frequency per 10 min to allow direct comparisons
between pre-/trial-/post-values. Several aggressive displays
and behavioral patterns were summed to produce one
composite data point for analysis; this high-level aggression category included the behaviors attack, chase, erh–
erh, cage mark, and piloerection [24,28]. A four-factor
analysis of variance was used to determine differences
among trial conditions, and LSD post hoc tests used to
evaluate all between-subjects main effects, and further
paired t-tests were used to analyze all interactions.
Specifically, for resident behaviors the design was a 2
(intruder sex)2 (intruder age)3 (time of the observation:
pretrial, during, post trial). A comparison was not made
comparing resident’s behavioral response to intruder
conditions versus the control condition because no
aggressive behaviors were displayed during any of the
trials in the presence of the empty cage. Intruder
demeanors and high-level aggressive behavior by the
intruder were analyzed using a 2 (intruder sex)2 (intruder
age)2 (trial condition: trial, control)3 (time of the
observation: pretrial, during, post trial) design.
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2.4.2. Hormone analysis
The hormonal data for both residents and intruders were
analyzed using the percent change relative to the baseline
morning samples, for the samples collected 2–6 h after the
trial (samples averaged together) and samples 24 h after the
trial, accounting for both short-term and long-term excretions. These times were chosen based on previous evidence
that excretion of CORT and T may commence as early as
2–6 h following a change in the plasma concentrations [30–
32]. Changes in T and CORT concentrations of male
residents were analyzed using an analysis of variance,
specifically 5 (exposure conditions: adult male intruder,
juvenile male intruder, adult female intruder, juvenile
female intruder, control)2 (time after the trial: 2–6 h, 24
h). The change in T and CORT for intruder males was
tested with a 2 (age of the intruder: adult, juvenile)2
(condition: intruder trial, control)2 (time after the trial: 2–
6 h, 24 h). The controls were included in the ANOVA in
order to determine whether changes in hormone concentrations throughout the day after an intruder trial were
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2.4. Data analyses
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2.3.3. Creatinine
All hormone concentrations were corrected for the
creatinine concentration of each sample to control for fluid
intake and output. Creatinine concentrations were measured
using a Jaffé end-point assay [33] previously described and
validated for C. kuhlii [29].
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2.2.4. Urine collection
Prior to testing (0600–0800 h), urine was collected
from all resident and intruder subjects using the procedures previously outlined in Ref. [29]. Urine was collected
every 2 h following the trial from each of the animals
tested until 1700 and then again the following morning
using positive reinforcement techniques. All samples were
centrifuged at 7000 rpm for 2 min to remove debris and
the supernatant was transferred to a clean minivial. The
samples were then stored at 20 8C until the assays were
performed.
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3.1.1. Behavioral measures
The occurrence of high-level aggressive behaviors
varied as a function of the interaction of the sex of the
resident and the sex of the intruder ( F(1,8)=10.5, P=0.01,
Fig. 1). Males reacted with a higher frequency of high-level
aggressive behavior during exposure to male intruders as
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compared to female intruders (male intruder: 3.71F0.77
displays/10 min; female intruder: 0.75F0.38 displays/10
min; P=0.02). Although residents showed lower rates of
agonistic behavior toward juvenile intruders relative to
adult intruders, the differences were not significant
( F(1,8)=3.4, n.s.). Male residents exhibited significantly
higher rates of genital displays during the trial and post-trial
observations than during the pre-trial observations
( F(2,8)=9.1, P=0.02, pre-trial: 0.09F0.01 displays/10
min; trial and post-trial: 1.16F0.68 displays/10 min). The
number of mating bouts significantly increased during and
following the trials, compared to the pretrial ( F(2,8)=17.49,
Pb0.001, pre-trial: 0.04F0.02 bouts/10 min; trial and posttrial: 0.34F0.32 bouts/10 min). No significant differences
across trial phases were found for vocalizations or scent
marking behavior.
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3.1.2. Testosterone
Male residents showed highly variable absolute concentrations of T, and the differences in mean T as a
function of intruder condition (adult male, juvenile male,
female, juvenile female, or control) were not significant
( F(1,4)=2.5, n.s.). Additionally, relative concentrations of
T, expressed as a proportion of pre-trial concentrations, did
not differ due to exposure to different intruder conditions
( F(1,4)=5.6, n.s.). However, the time after the intruder
encounter did affect the proportionate change in T concentration relative to pre-trial levels. Specifically, the change in
T from pre-trial was lower 2–6 h after the trial than 24 h
after the trial ( F(1,4)=8.38, P=0.04, 2–6 h: 5.85F3.67%;
24 h: 8.57F3.78%). This difference presumably reflects the
circadian rhythm of T [30].
While exposure to intruders was not associated with
consistent changes in average absolute or relative T
concentrations, the intensity of the aggressive interaction
in a given encounter with male intruders may serve as a
predictor of change in T subsequent to the encounter.
Resident males did not show a significant relationship
between high-level aggression and T prior to the trial or
changes in T 2–6 h or 24 h after the encounter when both
male and female intruders were included (prior: F(1,4)=
0.69, n.s.; 2–6 h: F(1,4)=1.49, n.s.; 24 h: F(1,4)=2.65, n.s.).
When the responses of male residents to male intruders were
analyzed several significant relationships were found. Highlevel aggression of a male resident towards a male intruder
was significantly related to T 2–6 h and 24 h following the
encounter (2–6 h: F(1,4)=7.57, P=0.05; 24 h: F(1,4)=9.02,
P=0.04). However, the aggression by the resident was not
related to T concentrations of the resident prior to the trial
( F(1,4)=1.68, n.s.). Fig. 2 indicates that residents tended to
show reduced T after trials involving fewer (0–10) highlevel aggression displays between residents and male
intruders, but large increases in T 24 h after trials
involving higher rates of high-level aggression and displays (11–40) (i.e.: attack, chase, erh–erh, piloerection, and
cage marking).
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distinct from changes that are associated with normal
circadian rhythms [31].
Paired t-tests or post-hoc LSD tests were used to explore
significant main effects and interaction effects revealed by
the ANOVA’s. One male resident provided a urine sample
that contained concentrations of T that were 15-fold higher
than his average hormonal concentrations (25,000 ng/mg
Cr, average=1607 ng/mg Cr). This sample was eliminated
and only concentrations from the second replicate trial were
used in the analysis for this male.
Unbalanced repeated measures SAS Proc Mixed was
used to compare the relationships between aggression and
the measured hormones, as well as examining the relationship between changes in T and CORT. For these analyses
the samples were not collapsed or averaged between trials.
Each resident was analyzed using every trial in which they
participated, i.e.: two adult males, and two adult females.
Hormone samples were averaged for the 2–6 h time period.
Analyses first examined the relationship between male
resident aggression and T or CORT for all intruders; further
analysis restricted the comparison to exposure to intruder
males only. Similar tests were used to compare changes in T
and CORT over time, as well as relationships between
aggression, T, and CORT for intruders. An a of 0.05 was set
for all analyses.
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Fig. 1. The number of high-level agonistic behaviors (including: attack,
chase, erh–erh, cage mark, and piloerection) displayed by male residents
during the intruder trials (intruder conditions: MA=male adult intruder,
MJ=male juvenile intruder, FA=female adult intruder, FJ=female juvenile
intruder). (Bars are meanFS.E.M., Pb0.05).
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F(1,4)=2.67, n.s.; 24 h: F(1,4)=1.81, n.s.). There were also
no significant relationships between the changes in hormone
concentrations (CORT 2–6/CORT 24: F(1,4)=0.64, n.s.;
CORT 2–6/T 2–6: F(1,4)=0.5, n.s.; CORT 2–6/T 24:
F(1,4)=1.33, n.s.; CORT 24/T 2–6: F(1,4)=0.55, n.s.; CORT
24/T 24: F(1,4)=0.8, n.s.; T 2–6/T 24: F(1,4)=3.01, n.s.).
We found no significant relationships between the
aggression displayed by the intruder and the aggression
displayed by the resident ( F(1,4)=0.5, n.s.). We also found
no significant relationships between the changes in residents’ hormones and the aggression displayed by the
intruder during the trial (resident T 2–6: F(1,4)=1.2, n.s.;
resident T 24: F(1,4)=0.5, n.s.; resident CORT 2–6:
F(1,4)=0.87, n.s.; resident CORT 24: F(1,4)=0.32, n.s.).
Finally, we found no significant relationships between the
intruder’s pretrial hormonal levels and the aggression
displayed by the resident during the trial (intruder T pre:
F(1,4)=0.08, n.s.; intruder CORT pre: F(1,4)=0.42, n.s.).
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3.2. Intruder responses
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3.2.1. Behavioral measures
Male and female intruders were equally aggressive
during trials ( F(2,12)=2.17, n.s.), and aggression was not
different among adult and juvenile intruders ( F(2,12)=1.32,
n.s.). As expected, during encounters with residents
intruders showed high rates of genital displays
( F(2,12)=4.2, P=0.04, trial: 0.09F0.01 displays/10 min;
pre- and post-trial: 0 displays/10 min) and twitters
( F(2,12)=4.93, P=0.05, trial: 0.3F0.2 twitters/10 min;
pre- and post-trial: 0.13F0.08 twitters/10 min). The
intruders scent marked significantly more during the posttrial observations than during the pre-trial or trial observations ( F(2,12)=5.37, P=0.02, post-trial: 6.69F2.75 scent
marks/10 min; pre-trial and trial: 1.22F0.65 scent marks/10
min). Adult intruders also scent marked significantly more
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3.1.3. Cortisol
Male residents did not have a differential change in
CORT concentrations after exposure to intruders ( F(1,4)=
2.6, n.s.). However, residents showed a significant change in
the concentration of CORT due to the time after the trial
( F(1,4)=7.8, P=0.04). The change in concentration 2–6 h
after the trial was significantly higher than the change in
concentrations 24 h after the trial (2–6 h: 34.34F11.5%;
24 h: 16.77F6.2%, Fig. 3). The control values represent the
daily circadian rhythm of CORT, and did not differ
significantly from the trial values.
Additionally there were no significant relationships
between aggression and CORT prior to, 2–6 h or 24 h after
the encounter with male and female intruders (prior:
F(1,4)=0.56, n.s.; 2–6 h: F(1,4)=0.81, NS; 24 h: F(1,4)=
0.82, n.s.). There were also no relationships between CORT
and T when intruders of both sexes were included (CORT
2–6/CORT 24: F(1,4)=2.78, n.s.; CORT 2–6/T 2–6:
F(1,4)=0.26, n.s.; CORT 2–6/T 24: F(1,4)=0.53, n.s.;
CORT 24/T 2–6: F(1,4)=0.79, n.s.; CORT 24/T 24:
F(1,4)=0.90, n.s.; T 2–6/T 24: F(1,4)=2.49, n.s.).
When the responses of residents to male intruders was
analyzed we found that aggression displayed by the resident
was not related to resident CORT prior to, 2–6 h or 24 h
following the encounter (prior: F(1,4)=0.07, n.s.; 2–6 h:
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Fig. 2. The relationship between the change in testosterone concentrations
2–6 h (top panel) and 24 h (bottom panel) after exposure to male adult and
juvenile intruders and number of high-level agonistic behaviors displayed
during the trial by the resident (n=15, Pb0.05).
Fig. 3. The percent change from baseline of cortisol concentrations for male
residents after intruder trials; there was a significant difference between
cortisol 2–6 h post and 24 h after the intruder trial for all conditions,
including the control (Pb0.05).
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Black tufted-ear marmoset males respond hormonally to
intruder challenges with increases in T if the encounter is
characterized by high levels of aggressive behavior. Both
immediately following the trial (2–6 h) and 24 h after
exposure to a male intruder, increases in T were positively
related to high levels of aggression, whereas a reduction or
no change in T was noted if the resident failed to respond
with aggressive displays. Variation in baseline concentrations of T in male residents was not associated with
variation in the amount of aggression displayed during the
encounter. This suggests that while T is responsive to
aggressive encounters, differences in baseline T do not
predict aggressive tendencies, at least in the context of a
resident male interacting with a male intruder. Therefore,
black tufted-ear marmosets exhibit physiological changes in
androgen concentrations that vary significantly as a function
of the resident’s own behavioral response to an intruder. In
contrast to T, levels of CORT in male residents were not
significantly altered as a consequence of the aggressive
interactions.
The change in T is related to the intensity of the
aggressive displays by the resident male, rather than the
presence of high agonism during the encounter. There is no
direct relationship between the intensity of the aggression of
the intruder and the change in the resident’s T. Additionally,
we found no direct relationship between the change in T for
the intruder and the intensity of the resident’s aggressive
displays. Therefore, it appears that the performance of
aggression, rather than the presence of or receipt of
aggression during an encounter, is associated with changes
in T.
The hormonal response of intruders during resident–
intruder encounters is very different from the response of the
resident. Intruders responded with few significant hormonal
changes following the encounter, although T did show
positive trends associated with aggression 2–6 h following a
trial. While residents showed an immediate (2–6 h) and long
lasting (24 h) change in T after the trial. Additionally, the
number of aggressive displays was substantially reduced in
intruders when compared to the frequency of aggression
displayed by residents (intruder: 0–6 displays/10 min;
resident: 0–40 displays/10 min). Therefore increases in T
in association with the amount of aggression performed
during the encounter differed based on the status of the
animal during the encounter, as seen with other mammals
[10–12,25,34,35].
It appears that the significant correlation between
aggression performed and changes in post-trial T concentrations is produced by two phenomena: (1) elevated T in
residents that behaved aggressively toward intruders, and
(2) reduced T in residents that did not behave aggressively
toward intruders. The first process suggests that engaging in
a competitive, agonistic interaction with a conspecific male
elevates androgen titers, perhaps facilitating the expression
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TE
3.2.2. Hormone response
Like male residents, mean levels of T and proportional
changes in T following the intruder encounter did not differ
for male intruders ( F(1,7)=4.5, n.s.). Male marmosets also
do not appear to have differential stress responses due to use
as an intruder. The change in CORT concentrations did not
differ due to the time after the trial, or the type of trial
( F(1,7)=2.6, n.s.).
An examination of T prior to the trial and changes in T 2–
6 and 24 h following an intruder trial revealed no relationships with aggression displayed by the intruder during the
trial (prior: F(1,6)=0.43, n.s.; 2–6 h: F(1,6)=0.13, n.s.; 24 h:
F(1,6)=0.01, n.s.) (Fig. 4). There was also no significant
relationship between aggression displayed during the trial
and CORT 2–6 and 24 h following the trial (2–6 h:
F(1,6)=0.68, n.s.; 24 h: F(1,6)=0.09, n.s.). However, CORT
prior to the trial was significantly related to the amount of
high level aggression displayed by the intruder during the
trial ( F(1,6)=8.97, P=0.04). While changes in T 2–6 h post
and 24 h post were related ( F(1,6)=6.26, P=0.05), there
were no other significant relationships between hormone
concentrations (CORT 2–6/CORT 24: F(1,6)=0.68, n.s.;
CORT 2–6/T 2–6: F(1,6)=0.08, n.s.; CORT 2–6/T 24:
F(1,6)=1.42, n.s.; CORT 24/T 2–6: F(1,6)=0.59, n.s.; CORT
24/T 24: F(1,6)=1.2, n.s.).
We found no significant relationships between the
intruder’s change in hormonal levels and the aggression
displayed by the resident towards the intruder (intruder T 2–
6: F(1,6)=1.1, n.s.; intruder T 24: F(1,6)=0.56, n.s.; intruder
CORT 2–6: F(1,6)=0.12, n.s.; intruder CORT 24:F(1,6)=
0.32, n.s.). There were no significant relationships between
aggression displayed by the intruder and pretrial levels of
residents’ hormones (resident T pre: F(1,6)=0.08, n.s.;
resident CORT pre: F(1,6)=0.49, n.s.).
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4. Discussion
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461 than did juvenile intruders ( F(1,7)=8.37, P=0.02, adult:
462 5.19F2.18 scent marks/10 min; juvenile: 0.9F0.5 scent
463 marks/10 min).
Fig. 4. The relationship between the change in testosterone concentrations
in intruder males 2–6 h (n=15), and 24 h after an intruder trial (n=15), and
high-level agonistic behaviors displayed by male intruders (n.s.).
7
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Acknowledgements
656
We would like to thank the following people for help in
data collection and animal management: Denise Hightower,
Danny Revers, Dan Jorgensen, Chad Hansen, Scott Nunes,
and Jeffrey Fite. We would also like to thank Jeff Fite,
Lauren Riters, and Erin Kinnally for helpful comments on
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have shown a direct increase in the concentrations of CORT
following an aggressive interaction such as one modeled by
our intruder trial [25,26,34] and these changes in CORT are
often associated with changes in T. However, an encounter
with intruders in marmosets does not appear to constitute a
significant psychosocial stressor, as indexed by HPA
function. The changes in CORT after an aggressive
encounter do not differ significantly from changes we
observed during the control conditions. The similarity of
changes in CORT excretion in control trials and intruder
trials suggests that we recorded differences attributable to
the circadian rhythm [31,32]. These results are similar to
those reported in the monogamous titi monkeys, who also
display little or no change in CORT following an encounter
with an unfamiliar conspecific [41]. Therefore, unlike
polygynous primates with low levels of paternal care, such
as the olive baboon [10] and squirrel monkey [41], it
appears that monogamous primates with a higher prevalence
of paternal care like the titi monkey and marmosets, do not
respond to intruder challenges with increases in concentrations of stress hormones. Additionally, our data reveal
that there is no direct relationship between CORT and T for
either the resident or intruder males. Therefore, it is unlikely
that differences in T concentrations following the intruder
encounter are associated with the suppression of gonadal
activity brought about by HPA activity.
In natural populations of marmosets, neighboring social
groups interact at the boundary of their territory on a regular
basis [18,19,42]. Extra-pair copulations have been noted
during some of the encounters, suggesting the possibility of
extra-pair fertilization in marmosets. Thus, the critical
preconditions favoring the elaboration of intermale aggression and its mediation or facilitation by increases in T are
clearly present in marmosets. At the same time, however,
elevated T may have a deleterious impact on male fitness.
We have demonstrated that the period of maximal paternal
effort by males, as indexed by infant carrying scores, is
associated with dramatic decreases in T concentrations [21].
Further, males that engage in high levels of paternal care
have significantly lower T, relative to males that engage in
low paternal effort [20]. The data we present in this paper
suggest that regulation of T titers in male marmosets is
exquisitely designed to reflect the competing demands of
intrasexual aggression and paternal care. Male marmosets
do in fact respond to agonistic challenges with increases in
T, but the magnitude of the response is conditional upon the
intensity of the encounter.
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of agonistic behavior in encounters in the near future. The
mechanisms and functions underlying the second process
are less clear. It has been demonstrated that engaging in
affiliative interactions does have an impact on endocrine
function, at least in the (HPA) axis [36,37]. We do not
necessarily suggest that a nonaggressive encounter with an
unfamiliar male constitutes an affiliative social interaction in
male marmosets, but it could be that T levels are reduced in
the aftermath of an interaction that is decidedly nonthreatening to the resident. The decreases in T following a
non-aggressive encounter may simply reflect the daily
circadian rhythm of T [30], or perhaps the male displaying
low levels of aggression may be the bloserQ of the encounter,
as seen in other primates [10–12]. In any event, this second
process deserves further attention.
The challenge hypothesis was proposed by Wingfield et
al. [4,38] to explain and predict changes in T due to
aggressive interactions as a function of the mating systems
of the birds. Specifically, in avian systems monogamous
males are predicted to show low basal levels of T during
times of paternal care, unless directly challenged, in which
case the T concentrations increase following the encounter.
However, polygynous males exhibit high levels of T
throughout the breeding season, and show no changes in
T following an encounter because concentrations are already
at a maximum. Although our data appears to support the
hypothesis that monogamous biparental mammals will show
increases in T due to an aggressive challenge, further testing
is needed. In order to explore whether T facilitates
aggression it is necessary to examine the responses of the
residents to future intruder encounters.
In all taxa, agonistic interactions among males vary
greatly in the intensity and duration of the interaction. A
host of factors contribute to this variation, including
behavioral and physiological predispositions of the participants. Tests of mammalian species have not specifically
examined the relationship between the amount of aggression
displayed by a resident and the testosterone concentrations.
Instead mammalian studies have focused on changes in
testosterone concentrations following either the presence or
absence of an encounter [39,40]. In many ways, the
paradigm we used with marmosets is a more dynamic one
than that typically tested in the avian or mammalian
systems. Marmosets were not screened prior to the tests
for aggressive tendencies; therefore both aggressive and
non-aggressive intruders were used in our encounters, which
produced considerable variability in the intensity of
aggression during encounters. Additionally, marmosets have
been shown in the past to have low levels of aggression
during encounters as compared to other Callitrichids [24].
This variability complicates the nature of the interactions,
but presumably our staged intruder encounters more closely
resemble variation in the intensity of actual encounters in
natural systems.
The interaction of conspecifics during an encounter is
often described as a stressful one [25,34]. Many studies
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662 the manuscript. Support for this project came from grants to
663 Jeffrey French from the National Science Foundation (IBN:
664 97-23842 and 00-91030).
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778
779
780
781
782
783
784
785
786
787
788